1. DNase H Activity of Neisseria meningitidis Cas9
Yan Zhang, Rakhi Rajan, H. Steven Seifert, Alfonso Mondragón, Erik J. Sontheimer 
Molecular Cell 
Volume 60, Issue 2, Pages (October 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 60, 242-255DOI: (10.1016/j.molcel.2015.09.020)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1 NmeCas9 Functions as an RNA-Guided DNA Endonuclease
(A) A Coomassie-stained 10% denaturing PAGE of wild-type and mutant NmeCas9 proteins. WT, wild-type NmeCas9; D16A, H588A, and D16A+H558A, active site NmeCas9 mutants D16A, H588A, and D16A+H558A. The predicted molecular weight of NmeCas9 is ∼110 kD. Weights of molecular markers are indicated.
(B) A schematic showing the complex of the sp 9 crRNA, the tracrRNA, and the target dsDNA. Yellow, crRNA spacer; gray, crRNA repeat; red, PAM; black arrows, predicted NmeCas9 cleavage sites.
(C) Plasmid cleavage by NmeCas9 requires tracrRNA, the cognate crRNA and Mg2+. N, nicked; L, linearized; SC, supercoiled.
(D) Sequencing of the NmeCas9 linearized pGCC2-ps 9 plasmid. Black arrows indicate predicted cleavage sites and the green stars indicate the A overhangs added by the sequencing reactions.
(E) Divalent metal ion specificity of NmeCas9. Plasmid cleavage assay was performed as in (C), except that dual RNAs were used for all lanes.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 Strand Specificity of NmeCas9’s Two Nuclease Domains
(A) Schematic representation of the location of the RuvC and HNH domains in the primary sequence of NmeCas9.
(B) Plasmid cleavage assays were performed using wild-type NmeCas9, as well as active site mutants D16A, H588A, and D16A+H588A. N, nicked; L, linearized; SC, supercoiled.
(C) NmeCas9 uses the HNH and RuvC nuclease domains to cleave the crRNA-complementary and noncomplementary strands, respectively. The oligonucleotide cleavage assay was performed using tracrRNA- and crRNA-programmed, wild-type and mutant NmeCas9 proteins at 37°C for 30 min. Duplex DNA substrates were 5′ 32P labeled on either strand. M, size markers. The sizes of the substrates, cleavage products and markers are indicated. The single and double asterisks denote noncomplementary strand and complementary strand (respectively) minor cleavage products that are abolished when either active site is mutated. The significance (if any) of these minor cleavage products is not clear.
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5. Figure 3 NmeCas9 Exhibits TracrRNA-Independent ssDNA Cleavage Activity
(A) Schematic representation of ps 25-containing DNA oligonucleotides and the sp 25 crRNA used in (B) and (C). Red lettering, PAM; yellow, crRNA spacer; gray, crRNA repeat; black arrows, predominant cleavage sites for dual-RNA guided NmeCas9 cleavage; red arrow, predominant cleavage site for tracrRNA-independent NmeCas9 cleavage.
(B) NmeCas9 cleaves ssDNA efficiently in a tracrRNA-independent manner. NmeCas9 complexed with small RNAs was assayed for cleavage of double- (left) or single- (right) stranded DNA targets for sp 25. The complementary strand was 5′ 32P radiolabeled. M, size markers. The sizes of substrates, cleavage products and markers are indicated.
(C) NmeCas9 binds an ssDNA target in vitro in a tracrRNA-independent manner. Gel shifts were performed using a 5′ FAM-labeled ssDNA substrate (100 nM), NmeCas9 (500 nM), and various small RNAs (500 nM) as indicated. Binding was performed in cleavage reaction buffer (but with Mg2+ omitted) at room temperature for 10 min, and then resolved by 5% native PAGE.
(D) An intact HNH domain is required for tracrRNA-independent cleavage of complementary strand ssDNA. Wild-type and active-site mutant NmeCas9s were assayed for cleavage of ssDNA, in the absence (left) or presence (right) of tracrRNA. Reactions were performed as in Figure 2C. M, size marker. The sizes of substrates, cleavage products and markers are indicated.
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6. Figure 4
Guide Sequence Independence and Cleavage Site Selection for NmeCas9’s DNase H Activity
(A) NmeCas9-catalyzed, crRNA-guided, tracrRNA-independent ssDNA cleavage does not require specific CRISPR repeats. Sp 25-specific crRNAs with repeat variants were assayed for NmeCas9-directed cleavage of an ssDNA target. Here and in the rest of this figure, cleavage reactions were performed as in Figure 2C, using a 5′ FAM-labeled ssDNA bearing a sp 25 target. The sizes of substrates and cleavage products are indicated. The panel of RNAs and substrates examined are depicted below the gel image and are colored as follows: DNA, brown lines and boxes with brown borders; RNA, black lines and boxes with black borders; ps 25, yellow; sp 25 sequences, red; sp 23 sequences, green; S. pyogenes (Sp) repeat, blue; 5′ labels, stars. The noncognate target is a 42 nt 5′ FAM-labeled ssDNA from the dTomato gene.
(B) ssDNA target cleavage by NmeCas9 is as efficient when crRNA is preannealed to DNA substrates (right), as compared to crRNA that is allowed to preload into NmeCas9 (left). Sp 25 crRNA (25, 50, 250, and 500 nM) is preannealed with ssDNA substrates (50 nM) in cleavage buffer at 37°C for 10 min. Preloading of crRNA (25, 50, 250, and 500 nM) with NmeCas9 (500 nM) is done in cleavage buffer at 37°C for 10 min.
(C) NmeCas9 determines the DNase H cleavage sites by a ruler mechanism that measures from the 5′ end of the DNA in the RNA-DNA hybrid, and requires a minimum 17–18 base-paired region. The guide-substrate pairs are depicted and named at the bottom of the panel. In the guides for samples 3–5, 7, and 8, additional nucleotides (complementary to the ssDNA target) were added to (or removed from) either the 5′ or 3′ end of the guide, with the specific end and the number of added or subtracted nucleotides indicated. All RNA guides have a 5′-terminal GGG trinucleotide to facilitate in vitro transcription, so the lengths of the DNA-RNA hybrid duplexes are 3 nt shorter than the RNA guides.
(D) Inverting the backbone composition of the guide-substrate duplex also inverts the strand asymmetry of the cleavage activity. 1′, 2′, and 3′ denote the control reactions including only the labeled DNA component for substrate pairs 1, 2, and 3, respectively. The basis for the slightly retarded mobility of the labeled target upon incubation with crDNA (substrate pair 2) is not known.
(E) DNase H activity of apo NmeCas9. The depiction of NmeCas9 is based on the structure of a different Type II-C Cas9 (AnaCas9; Jinek et al., 2014). Recognition lobe, dark gray; nuclease lobe, light gray; HNH domain, yellow.
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7. Figure 5 NmeCas9 Functions with a Range of PAM Variants
(A) NmeCas9 cleavage of plasmids containing ps 9 and wild-type or mutant PAMs. NmeCas9 was programmed with tracrRNA and the sp 9 crRNA. Reactions were performed as in Figure 1C. Mutations in the PAM are indicated in red, and PAMs are in bold.
(B) NmeCas9 targets a range of PAM variants in a cellular interference assay. Plasmids containing ps 25 and various PAM mutants were tested by natural transformation assays using wild-type MC8013 cells as recipients. The bar graphs are log-scale plots of colony-forming units (CFU)/ml (mean ± SEM) for total cells (blue bars) and erythromycin-resistant (ErmR) transformants (red bars) from three independent experiments. The positions and nucleotide identities of the mutations are indicated for each PAM variant.
(C) PAM residues are required on both strands to license NmeCas9 cleavage on a duplex DNA substrate. NmeCas9 programmed with tracrRNA and sp 25 crRNA were tested for oligonucleotide cleavage as in Figure 2C. Non-C, noncomplementary strand; C, complementary strand. Duplex DNA oligos were 5′ 32P-labeled on both strands. Mutant PAM sequences are shown (below the gel) in red, with the PAM region in bold.
(D) NmeCas9 cleavage of ssDNA targets does not require a PAM. The oligonucleotide cleavage assay was performed as in Figure 2C, except that 5′ FAM-labeled complementary ssDNA oligos (100 nM) were used. NmeCas9 was programmed either with sp 25 crRNA alone or together with tracrRNA. PAM mutations are indicated (below the gel) in red, and PAMs are in bold. The “No PAM” oligonucleotide carries a triple mutation in the PAM.
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8. Figure 6
NmeCas9 Has Minimal Tolerance for Protospacer-PAM Linker Length Variation
(A) NmeCas9 only functions with a 4 bp linker in bacterial cells. Left, cellular interference assay. Plasmids containing ps 23 and its linker length mutant derivatives were tested in natural transformation assays as in Figure 5B. Sequences of the linkers between ps 23 and its PAM in the mutants used in (A) are shown on the right. The linkers are in red.
(B) The same plasmids tested in (A) were assayed for cleavage in vitro by NmeCas9 programmed with sp 23 crRNA and tracrRNA. Reactions were performed as in Figure 1C. N, nicked; L, linearized; SC, supercoiled.
(C) NmeCas9 cleavage of the noncomplementary strand in DNA duplexes is much less efficient when the linker length varies, and the cut site moves in concert with the PAM. The duplex DNAs were 5′ 32P labeled on the noncomplementary strand only, and contain ps 25, a flanking GATT PAM and a 2–6 nt linker in between. NmeCas9 was programmed with tracrRNA and sp 25 crRNA. Reactions were performed as in Figure 2C. M, size markers. The sizes of substrates, cleavage products and markers are indicated.
(D) NmeCas9 cleavage of the complementary strand ssDNA is as efficient when linker length varies, and the cut sites are at a fixed position. The ssDNA targets were 5′ 32P labeled, and contain ps 25, a flanking GATT PAM, and a 2–6 nt linker in between. NmeCas9 was programmed with sp 25 crRNA and with or without tracrRNA, as indicated. Reactions were performed as in Figure 2C. M, size markers. The sizes of substrates, cleavage products and markers are indicated.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7 NmeCas9 sgRNA Requirements In Vitro and in Bacteria
(A) A schematic of the NmeCas9 sgRNA, a chimeric fusion in which the 3′ end of the mature crRNA and the 5′ end of the mature tracrRNA are connected by a GAAA tetraloop. Highlighted in yellow, crRNA spacer; highlighted in gray, crRNA repeat. Six 3′ terminal truncations are indicated by red arrows, and three internal deletions are indicated by colored boxes.
(B) Full-length and variant sgRNAs were assayed for NmeCas9 cleavage of ps 9-containing plasmid in vitro. NmeCas9 was programmed with tracrRNA and sp 9 crRNA. Reactions were performed as in Figure 1C. The full-length tracrRNA used in in vitro assays had 6 additional 3′-terminal nucleotides (compared to the sequence shown in A), whereas all the other RNAs are as shown. N, nicked; L, linearized; SC: supercoiled.
(C) Schematics of representative MC8013 isogenic strains used in (D). Relevant genotypes are given on the left. Grey boxes, genes; blue boxes, kanamycin-resistance marker; black rectangles, CRISPR repeats; white diamonds, CRISPR spacers; green boxes, tracrRNA promoter.
(D) Definition of sgRNA regions that are required for transformation interference. pYZEJS040 (−) and its ps 25-containing derivative (+) were introduced into MC8013-derived strains by natural transformation. Relevant genotypes and sgRNA complementation variants are indicated at the bottom. Data are presented as in Figure 5B.
Molecular Cell  , DOI: ( /j.molcel )
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